Foreword by Ernst H. K. Stelzer

In light sheet-based fluorescence microscopy (LSFM), optical sectioning in the excitation process minimizes fluorophore bleaching and phototoxic effects. This allows biological specimens to survive long-term three-dimensional multi-spectral imaging at high spatiotemporal resolution along multiple directions. The illumination of an entire plane allows the recording of images with a camera. Hence, millions of pixels are recorded in parallel, while several tens or hundreds of image are recorded within a few seconds.

LSFM has revolutionized fluorescence microscopy as it allows scientists to perform experiments in an entirely different manner and to record data that had not been accessible before. LSFM is a disruptive technology, because it forces the scientific community to re-think the manner in which it performs its experiments, to avoid flat and hard surfaces, to reconsider quality criteria, and to study as well as maintain the three-dimensional dynamic multicellular structure of essentially all biological specimens.

LSFM is based on an extremely simple and yet ingenious optical arrangement that provides true optical sectioning over an extended field of view. In contrast to epi-fluorescence microscopy, LSFM refers to a technology that observes a specimen with at least one microscope objective lens whose focal plane is illuminated azimuthally by at least one sheet of light. In LSFM, only a thin volume wrapped around the focal plane of the detection microscope objective lens is illuminated. Therefore, endogenous organic molecules or fluorophores in the volumes in front and behind the light sheet do not receive any light and are not subject to photo-damage. These fluorophores cannot contribute to image blurring by out-of-focus light. Three-dimensional image stacks are generated by moving the light sheet and the specimen relative to each other.

In physical terms, LSFM provides true optical sectioning and, therefore, a three-dimensional resolution. Hundreds of planes in different locations along the optical axis of the detection lens are independently illuminated. Since, in to contrast to confocal fluorescence microscopy, the illumination process provides the optical sectioning capability of LSFM, it exposes specimens to 3-5 orders of magnitude less energy than confocal microscopy.

In general, optical sectioning and no phototoxic damage or photo bleaching outside a small volume close to the focal plane are intrinsic properties of LSFM. The two canonic implementations are selective/single plane illumination microscopy (SPIM) [1] and digital-scanned laser light sheet microscopy (DSLM) [2], which provide a coherent and an incoherent illumination, respectively. In particular, DSLM has become an indispensable tool in developmental biology, three-dimensional cell biology, and plant biology as well as for cleared and expanded specimens. DSLM is also the basis for most LSFM-derived technologies that try to improve spatial resolution.

Light sheets have been known for more than 100?years, but so have light spots. However, until lasers became available in 1960 [3], neither light spots nor light sheets were diffraction limited. Hence, optically sectioning instruments [4] could not be built. A confocal fluorescence microscope, which illuminates a specimen sequentially with a diffraction-limited spot of light, requires a laser as its light source. LSFM, which illuminates a specimen sequentially plane by plane with a diffraction-limited light sheet, also requires a laser as its light source.

Laser light sheet-based macroscopic devices [5] had been built several times, but their capability to perform at a microscopic level was not known until, starting around 2002, my group (Light Microscopy Group [LMG]), then at the European Molecular Biology Laboratory (EMBL) in Heidelberg, built, patented, and applied the SPIM. LSFM was used to observe live biological specimens, assessed for its optical properties [6], evaluated for its applicability for multiple-views imaging (7, 8] and considered for its applicability at the molecular level with fluorescence lifetime imaging microscopy (FLIM)/fluorescence resonance energy transfer (FRET) [9] as well as fluorescence correlation spectroscopy (FCS)-selective plane illumination microscopy (SPIM) [10]. EMBL's LMG had systematically evaluated diffraction-limited microscopes with two to four lenses, both in theory and in practice, since the early 1990s. The disruptive impact of LSFM was recognized in 2015, when Nature Methods announced "Light Sheet-based Fluorescence Microscopy" Method of the Year 2014 [11].

LSFM is the result of developing confocal [12], 4Pi [13], and Theta [14] fluorescence microscopies for about 20?years. It is an excellent example for the achievements of scientists with an interdisciplinary spirit. Patents and papers published since 1993 and seminal papers published since 2004 document the intensity and the determination that are necessary to make the scientific community aware of a disruptive technology. Much further work and the refinements of LSFM enable the imaging of live biological samples under close-to-natural conditions for several days, leading to breakthroughs in, among other fields, developmental biology, neurobiology, and histopathology as well as drug development. The initial work was performed at EMBL. However, many brilliant people have independently pushed the technology to new levels all around the world.

My scientific profile describes a physicist who worked in interdisciplinary environments for more than 35 years. I was able to bridge gaps between physics, optics, and instrumentation on one side and molecular, cell, plant, and developmental biology on the other side. The many steps that are required for reasonable biological systems, excellent data and well-defined mathematical-physical interpretations of biological processes have guided me in my decisions during many seemingly different projects.

During my PhD thesis (1983-1987), I worked on confocal transmission, reflection, and fluorescence microscopy. I developed confocal 4Pi fluorescence microscopy during 1990-1993 and introduced orthogonal and multi-lens detection schemes commencing with confocal theta fluorescence microscopy around 1993. The latter led to the development of the tetrahedral microscope in 1999, which in turn triggered the development of LSFM in 2001. Some of my other contributions include the optical tweezers-based photonic force microscope in 1993 and a novel and very successful approach to laser-based cutting devices in 1999.

I am extremely grateful that I had, and still have, the opportunity to work with many brilliant people who have contributed enormously to the topics that I mentioned before. I seriously hope that I inspire them because I can assure you that they certainly inspired me.

December 2019

Ernst H. K. Stelzer,
Goethe-Universität, Frankfurt am Main, Germany

References

1. 1 Huisken, J., Swoger, J., Del Bene, F. et al. (2004). Optical sectioning deep inside live embryos by selective plane illumination microscopy. Science 305: 1007-1009.
2. 2 Keller, P.J., Schmidt, A.D., Wittbrodt, J., and Stelzer, E.H.K. (2008). Reconstruction of zebrafish early embryonic development by scanned light sheet microscopy. Science 322: 1065-1069.
3. 3 Maiman, T.H. (1960). Stimulated optical radiation in ruby. Nature 187: 493-494.
4. 4 Cox, I.J. (1984). Scanning optical fluorescence microscopy. Journal of Microscopy 133: 149-154.
5. 5 Voie, A.H., Burns, D.H., and Spelman, F.A. (1993). Orthogonal-plane fluorescence optical sectioning: three-dimensional imaging of macroscopic biological specimens. Journal of Microscopy 170: 229-236.
6. 6 Engelbrecht, C.J. and Stelzer, E.H.K. (2006). Resolution enhancement in a light-sheet-based microscope (SPIM). Optics Letters 31: 1477-1479.
7. 7 Swoger, J., Verveer, P., Greger, K. et al. (2007). Multi-view image fusion improves resolution in three-dimensional microscopy. Optics Express 15: 8029-8042.
8. 8 Verveer, P.J., Swoger, J., Pampaloni, F. et al. (2007). High-resolution three-dimensional imaging of large specimens with light sheet-based microscopy. Nature Methods 4: 311-313.
9. 9 Greger, K., Neetz, M.J., Reynaud, E.G., and Stelzer, E.H.K. (2011). Three-dimensional fluorescence lifetime imaging with a single plane illumination microscope provides an improved signal to noise ratio. Optics Express 19: 20743-20750.
10. 10 Wohland, T., Shi, X., Sankaran, J., and Stelzer, E.H.K. (2010). Single plane illumination fluorescence correlation spectroscopy (SPIM-FCS) probes inhomogeneous three-dimensional environments. Optics Express 18: 10627-10641.
11. 11 Stelzer, E.H.K. (2015). Light-sheet fluorescence microscopy for quantitative biology. Nature Methods 12: 23-26.
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